The long-term goal of this project is to elucidate the cellular, molecular, and biochemical mechanisms regulating the proliferation and motility of human uterine smooth muscle cells (UtSMC). UtSMC hyperproliferation is the cause of fibroids, a condition that afflicts 20-25% of all women and 75% of African-American women. Fibroids cause severe pain and bleeding, impair fertility, and result in >200,000 hysterectomies annually in the U.S. There is no known treatment--medical or surgical--that permanently reduces or eliminates fibroids, other than hysterectomy. Clearly, a detailed understanding of the mechanisms and molecules that regulate UtSMC mitogenesis and migration will provide a therapeutic rationale for controlling fibroids, and may provide important insights into the pathophysiologic basis for fibroid formation. Our laboratory has provided strong evidence that CCN5, an estrogen-induced growth-arrest specific gene, inhibits proliferation and motility in cultured UtSMC. Furthermore, we have demonstrated that human leiomyomas have greatly reduced levels of CCN5 mRNA and protein compared to normal myometrium from the same uterus. Based on this evidence the following hypothesis will be tested: CCN5 is an autocrine regulator of UtSMC proliferation and motility in culture and in vivo, and exerts it anti-proliferative and anti-motility effects, at least in part, through regulation of extracellular matrix synthesis and composition. To test this hypothesis, we will: 1) Continue our functional analysis of CCN5 and its regulation by estrogen on proliferation, motility, and extracellular matrix in SMC cultured from matched pairs of normal and fibroid human uterine tissue. To do this we will use adenovirus vectors, recombinant CCN5, and small inhibitory RNA approaches. 2) Examine the physiologic functions and estrogen regulation of CCN5 in animal models, including normal cycling rats, ovariectomized rats, pregnant rats, wild-type mice, and genetically manipulated mice that either under- or over-express CCN5. Quantitative PCR, Western blot analysis, and immunohistochemistry will be used to determine the spatial and temporal expression pattern and estrogen regulation of CCN5 in each of these animal models. We will also explore the possibility that CCN5 gene or protein therapy might be a useful approach for suppressing human fibroids in a novel nude mouse model system. The experiments proposed in this application should provide new and important insights into UtSMC pathophysiology in humans.